The Caenorhabditis elegans ARIP-4 DNA helicase couples mitochondrial surveillance to immune, detoxification, and antiviral pathways

Significance Eukaryotes surveil and respond to mitochondrial dysfunction caused by toxins or mutations, triggering antiviral and immune responses as well as mitochondrial repair. A mutation in the Caenorhabditis elegans mitochondrial chaperone hsp-6 activates detoxification and RNA interference defense pathways. Mitochondrial mutations increase C. elegans longevity, and mutations that disable RNA interference suppress this longevity increase. Mitochondria also serve as a signaling hub for mammalian immune responses to viruses. The conserved DNA helicase gene arip-4 is required for the increase in antiviral RNAi caused by mitochondrial dysfunction. But an arip-4 mutation extends the life span of the hsp-6 mitochondrial mutant, suggesting that the futile activation of detoxification pathways is deleterious to health when the mitochondrial dysfunction is caused by mutations.

Surveillance of Caenorhabditis elegans mitochondrial status is coupled to defense responses such as drug detoxification, immunity, antiviral RNA interference (RNAi), and regulation of life span. A cytochrome p540 detoxification gene, cyp-14A4, is specifically activated by mitochondrial dysfunction. The nuclear hormone receptor NHR-45 and the transcriptional Mediator component MDT-15/MED15 are required for the transcriptional activation of cyp-14A4 by mitochondrial mutations, gene inactivations, or toxins. A genetic screen for mutations that fail to activate this cytochrome p450 gene upon drug or mutation-induced mitochondrial dysfunction identified a DNA helicase ARIP-4 that functions in concert with the NHR-45 transcriptional regulatory cascade. In response to mitochondrial dysfunction, ARIP-4 and NHR-45 protein interaction is enhanced, and they relocalize from the nuclear periphery to the interior of intestinal nuclei. NHR-45/ARIP-4 also regulates the transcriptional activation of the eol-1 gene that encodes a decapping enzyme required for enhanced RNAi and transgene silencing of mitochondrial mutants. In the absence of arip-4, animals were more susceptible to the mitochondrial inhibitor antimycin. Thus, ARIP-4 serves as a transcriptional coactivator of NHR-45 to promote this defense response. A null mutation in arip-4 extends the life span and health span of both wild type and a mitochondrial mutant, suggesting that the activation of detoxification pathways is deleterious to health when the mitochondrial dysfunction is caused by mutation that cannot be cytochrome p450-detoxified. Thus, arip-4 acts in a pathway that couples mitochondrial surveillance to the activation of downstream immunity, detoxification, and RNAi responses.

mitochondria | RNAi | detoxification | healthspan
Human aging is accompanied by diverse pathologies, including cancer, diabetes, neurodegeneration, and cardiovascular diseases, suggesting a complex and progressive decay of multiple pathways (1). A decline in cellular protection, homeostasis, and regeneration contributes to senescence (2). Despite this complexity, genetic analyses have revealed single-gene mutations that can extend life span; such mutations may trigger life spanprolonging genetic programs that are normally triggered by nutritional or pathogen cues. One of the largest class of mutations that increase longevity in the nematode Caenorhabditis elegans are mutations that reduce mitochondrial function (3,4).
The mitochondrion has its origin from an ancient endosymbiotic association with an alpha-proteobacteria, and most of the few thousand bacterial genes of the original endosymbiont migrated to the nucleus where these genes are regulated no differently from other nuclear genes; just a few dozen genes remain in the mitochondrial genomes of eukaryotes. But the proteins encoded by the nuclear genes that encode mitochondrial proteins retain many of their functions for metabolism and electron transport, for example, and are localized to the mitochondria after translation on the cytoplasmic and secretory ribosomes. More than 1,000 nuclear-encoded proteins with a deep bacterial ancestry reassemble the mitochondrion from its nuclear diaspora bacterial ancestral genome in each eukaryotic cell (5). A large fraction of the nuclear encoded mitochondrial proteins retain homology to modern bacterial proteins. Most bacterial species and most eukaryotic mitochondria generate energy via proton pumping across the mitochondrial membranes by the many heme and iron-sulfur proteins of the electron transport chain (ETC). The free energy of this ETC-generated proton gradient drives the synthesis of adenosine triphosphate (ATP) by the F 1 F 0 -ATP synthase to power thousands of enzymatic reactions. The bacterial origin of the eukaryotic mitochondrion allows the highly evolved weapons of 4 billion years of bacterial competition between each other to be marshaled by bacterial pathogens against eukaryotes. Because iron became a limiting factor in many ecosystems after the evolution of bacterial photosynthesis, the mitochondrion is an attractive target of bacterial pathogens that may find the bacterial lineage of mitochondria familiar and vulnerable to their highly evolved antibacterial virulence weapons (6,7).
The mitochondria also serve as a key signaling hub for immune responses to viruses. For example, the mammalian mitochondrial antiviral signaling protein (MAVS) associates with viral RNA sensor retinoic acid-inducible gene I (RIG-I) or melanoma differentiationassociated protein 5 (MDA5) to trigger the downstream nuclear factor kappa B, as well as other interferon immune signaling (8). In addition, the mitochondrion harbors its own idiosyncratic genome encoding just 13 protein-coding genes, 22 tRNA genes, and two ribosomal RNA genes. DNA or RNA released from the mitochondria may share more features with the nucleic acids of viruses than the nuclear genes, and is interpreted as "foreign," eliciting immune responses through RIG-I or MDA5 and MAVS (9,10).
RNA interference (RNAi) is highly conserved across most eukaryotes. It deploys 22-to 26-nt single-stranded small interfering RNAs (siRNAs) produced by Argonaute proteins and the Dicer dsRNA ribonuclease, as well as RNA-dependent RNA polymerases in a large fraction of eukaryotes but in a small fraction of animal genomes (11). These siRNAs target mRNAs for degradation or chromatin regions for epigenetic silencing across most eukaryotes (11). RNAi was first identified in C. elegans and plants by its antiviral activities that detect and cleave foreign nucleic acids such as viruses (12,13). The C. elegans RNA helicase DRH-1 that is orthologous to mammalian RIG-I or MDA5 regulates antiviral RNAi responses to degrade invading viruses (14,15). In mammalian cells, release of mRNAs transcribed from the mitochondrial genome into the cytosol is an immune elicitor (16); similar activation of immune response by mitochondrial RNA release has been observed in Drosophila melanogaster and C. elegans (17,18). In C. elegans, mitochondrial dysfunction due to a mutation in the mitochondrial chaperone gene hsp-6 causes an enhanced level of RNAi and transgene silencing that is a classic feature of the induction of antiviral RNAi (18,19).
Over the past two billion years, eukaryotes have evolved signaling networks to detect mitochondrial dysfunction, caused either by toxins from pathogens or by mutations that can be misconstrued by toxin or pathogen surveillance pathways as a pathogen attack, to trigger defense of their mitochondria. In C. elegans, the mitochondrial unfolded protein response (UPR mt ) is a mitochondria-to-nuclear communication channel that is activated by various types of mitochondrial dysfunction, including natural toxins such as oligomycin or mutations in mitochondrially localized nuclear-encoded proteins (20). The transcriptional outputs of UPR mt include mitochondrial chaperones, proteases, detoxifying enzymes, and secreted antibacterial proteins that enable defense and recovery of mitochondrial function. Detection of C. elegans mitochondrial dysfunction couples to the coordinated induction of drug detoxification and immune response genes (21)(22)(23). We previously identified a nuclear hormone receptor (NHR) NHR-45 and a Mediator component MDT-15/MED15 as components of a dedicated transcriptional pathway for mitochondrial dysfunction responses (23).
Here, we report the identification of the conserved DNA helicase RAD-26/ARIP-4 in a large genetic screen for mutants that fail to activate detoxification response to mitochondrial dysfunction. arip-4 is required for the activation of the cytochrome p450 xenobiotic detoxification gene cyp-14A4, normally activated by mitochondrial dysfunction. We find that the ARIP-4 DNA helicase associates with the NHR-45 to transcriptionally activate drug detoxification and immunity genes and mediate resistance to the mitochondrial inhibitor antimycin or pathogenic bacteria Pseudomonas aeruginosa. The NHR-45/ARIP-4 pathway also regulates the increase in antiviral RNAi response caused by mitochondrial dysfunction through induction of the decapping enzyme gene eol-1. A null mutation in arip-4 extends the life span and the health span of both wild type and a mitochondrial mutant, suggesting that the activation of detoxification pathways is deleterious to health when the mitochondrial dysfunction is caused by mutation that cannot be cytochrome p450-detoxified rather than by a toxin.

Activation of the Xenobiotic Reporter cyp-14A4p::gfp Requires
RAD-26/ARIP-4. Surveillance of mitochondrial integrity is essential for maintaining cellular homeostasis and is coupled to a complex signaling network that induces mitochondrial recovery, drug detoxification, and immunity. Although almost all of the known mitochondrial stress-related transcriptional responses required the UPR mt , certain responses, such as detoxification and immunity, needed additional factors (24). There are about 250 C. elegans drug detoxification genes of four major classes: cytochrome p450, uridine diphosphate (UDP) glycosylating, sulfate and glutathione S-transferase (GST) genes that add hydrophilic groups to generally hydrophobic toxins, and ABC transporters that excrete the modified toxins (25). Different suites of these detoxification genes are triggered by mitochondrial dysfunction compared with those that are induced by ribosomal dysfunction or cytoskeletal dysfunction, for example (21). The many drug detoxification genes have evolved by gene duplication and divergence and by the acquisition of promoters that are activated by distinct cellular challenges. cyp-14A4 is one of about 77 C. elegans cytochrome p450 genes and is induced specifically by a variety of mitochondrial mutations, gene inactivations, or toxins but not, for example, by ribosomal dysfunction or cytoskeletal dysfunction (21,25). A fusion of the cyp-14A4 promoter to GFP, cyp-14A4p::gfp, is induced in the intestine by a variety of mitochondrial mutations (23). For example, cyp-14A4p::gfp is induced by hsp-6(mg585), a hypomorphic mutation in the mitochondrial chaperone HSP-6/mtHSP70.
A genome-wide ethyl methanesulfonate (EMS) mutagenesis screen for mutants that fail to induce cyp-14A4p::gfp in hsp-6(mg585) revealed an allele mg691, a Trp371 to Stop (TGG to TAG) nonsense mutation in rad-26 ( Fig. 1 A and B). After an EMS mutagenesis, any F2 mutant selected for any phenotype carries 100-300 distinct mutations distributed across its genome; any one of those mutations could cause the new phenotype, in this screen, failure to induce cyp-14A4p::gfp in the hsp-6(mg585) mitochondrial mutant. However, many of the EMS mutations will be located in introns or intergenic regions, and many are not predicted to cause amino acid substitutions (26). After the detection of a candidate lesion such as rad- 26(mg691), it is necessary to reconstruct a similar mutation in the absence of the other EMS lesions traditionally by multiple backcrossing but recently using the CRISPR to engineer only that single lesion in an unmutagenized background.
To establish that the lesion in rad-26 in this F2 mutant isolated after a mutagenesis screen was the causative mutation for the failure to induce cyp-14A4p::gfp in hsp-6(mg585), CRISPR-Cas9 editing was used to generate just a single independent null allele of rad-26(mg697) with 28 base pairs inserted that caused a frameshift (Fig. 1B). The loss-of-function rad-26(mg697) mutant also disrupted induction of cyp-14A4p::gfp in the hsp-6(mg585) background ( Fig. 1 C and D). The rad-26 requirement for the activation of cyp-14A4 expression was not limited to the hsp-6(mg585) mitochondrial mutant: The complex III natural toxin antimycin isolated from Streptomyces bacteria also activates cyp-14A4p::gfp, and this activation is dependent on rad-26 gene activity ( Fig. 1 C and D). The human pathogenic bacteria P. aeruginosa produces chemical toxins that disrupt the mitochondrial membrane potential and virulence factors that inhibit translation (27,28). P. aeruginosa also activates cyp-14A4p::gfp ( Fig. 1 C and D), and this induction is dependent on rad-26 ( Fig. 1 C and D). Thus, rad-26 is necessary for the coupling of mitochondrial dysfunction caused by mitochondrial mutations or toxins to the activation of the detoxification gene cyp-14A4.
The 1,274 amino acid proteins encoded by rad-26 is one of 20 paralogous DNA helicases in the C. elegans genome. These DNA unwinding helicases hydrolyze ATP to supply free energy to break the hydrogen bonds between annealed nucleotide bases and separate double-stranded DNA into single strands (29). There are 31 DNA helicases in the human genome, classified into six groups (30). Although the C. elegans nomenclature RAD-26 refers to Saccharomyces cerevisiae Rad26 (RADiation sensitive), these two proteins are not orthologs but paralogs. They are distant from each other on the phylogenetic tree of these DNA helicases (Fig. 1E). S. cerevisiae Rad26 is responsible for transcription-coupled nucleotide excision repair (31), and its ortholog is ERCC6 in human and CSB-1 in C. elegans (Fig. 1E). The phylogenetic tree shows that C. elegans rad-26 encodes a conserved Snf2-like DNAdependent ATPase orthologous to human ARIP4 (androgen receptor-interacting protein) (Fig. 1E). Because there is essentially no literature about the genetic function of the C. elegans rad-26 gene and because it is an ortholog of mammalian ARIP4, both based on the phylogenetic tree and based on its function with NHRs (see below), we rename this C. elegans gene arip-4.
The Snf2 family DNA helicases are involved in transcription regulation, chromatin remodeling, homologous recombination, and DNA repair (32). When participating in transcription, these DNA helicases function as coregulators and interact with transcriptional factors, including NHRs. Fitting with the cyp-14A4 transcriptional regulatory function of C. elegans arip-4, and with its genetic and biochemical interaction with the C. elegans NHR NHR-45 (see below), human ARIP4 is a transcriptional coregulator for NHRs (33,34). ARIP4 belongs to the Snf2 family DNA helicases and serves as a transcriptional coactivator to enhance androgen-dependent transcriptional programs (35). ARIP4 may be animal specific ( Fig. 1E) because no ortholog exists in S. cerevisiae or Arabidopsis thaliana (32). Loss of ARIP4 in mice is embryonic lethal with increased apoptosis and decreased DNA synthesis (36).

C. elegans ARIP-4 Serves as a Transcriptional Coactivator for the NHR NHR-45 that Acts in the Mitochondrial Dysfunction
Detoxification Response Pathway. The C. elegans NHR NHR-45 and Mediator subunit MDT-15 are required for coupling the detection of mitochondrial dysfunction to the upregulation of the cyp-14A4 detoxification gene (23). Because the mammalian ortholog of C. elegans ARIP-4, ARIP4, directly interacts with NHR proteins, we hypothesized that C. elegans ARIP-4 may associate with NHR-45 to regulate cyp-14A4 transcription.
We first tested whether C. elegans ARIP-4 is localized to the nucleus. In mammals, the majority of detoxifying cytochrome p450 are expressed in the liver; in C. elegans, there is no liver, but the 32 intestinal cells may perform both gut and liver functions, such as detoxification (37). The gut is also where C. elegans would be expected to encounter bacterial pathogens that disrupt mitochondrial function and where immune responses to bacterial toxins and virulence factors would be expected. Consistent with the homology between the C. elegans gut and mammalian liver, cyp-14A4p::gfp is predominantly induced in the intestine upon mitochondrial dysfunction in all cell types (23). To observe C. elegans ARIP-4 protein localization, GFP was fused at the poorly conserved C terminus of ARIP-4 under the control of an intestine-specific promoter vha-6p in a miniMOS vector to generate a single-copy transgene vha-6p::arip-4::gfp::tbb-2 3'UTR fusion protein (38). The protein is localized to the nucleus in the intestine, where the vha-6 promoter drives the expression of the ARIP-4::GFP fusion protein ( Fig. 2A). Interestingly, although the ARIP-4::GFP fusion protein localized in the intestinal nucleus in both wild-type and hsp-6(mg585) animals, the intranuclear patterns were different ( Fig. 2A). In the wild-type nucleus, the ARIP-4::GFP fusion protein localized to the nuclear periphery, but in the hsp-6(mg585) mitochondrial mutant, ARIP-4::GFP was localized to the nuclear interior ( Fig. 2A).
We hypothesized that ARIP-4 relocalization to the nuclear interior may depend on the localization of an interacting transcription factor, for example, NHR-45. We tested whether the ARIP-4 and NHR-45 proteins localize to the same region of intestinal nuclei in wild-type or mitochondrial mutant animals. To be compatible with the ARIP-4::GFP fusion protein, NHR-45 was tagged with mScarlet at its C terminus using the intestine-specific vha-6 promoter to drive expression. In the hsp-6(mg585) mitochondrial mutant, NHR-45::mScarlet showed a similar shift of localization away from the nuclear periphery (Fig. 2B). This change of intranuclear position is consistent with models of chromatin states: Closed chromatin with repressed transcription tends to reside at the nuclear periphery, and open chromatin with active transcription tends to be more intranuclear (39).
Not only do ARIP-4 and NHR-45 colocalize and coregulate cyp-14A4, ARIP-4 protein physically interacts with the NHR-45 protein in a similar manner to that of its human ortholog ARIP4, which binds to another NHR protein, the androgen receptor (35). To detect this interaction, NHR-45 was fused with FLAG tag at its C terminus driven by the vha-6 promoter. Complexes from protein extracts of C. elegans were first immunoprecipitated using the tag on ARIP-4::GFP and tested for coimmunoprecipitation of NHR-45::FLAG. The two proteins interacted in wild-type animals, but the interaction was enhanced in the hsp-6(mg585) mitochondrial mutant (Fig. 2C). mRNA-seq analyses of hsp-6(mg585) and hsp-6(mg585); nhr-45(mg641) mutants (23) revealed that a variety of detoxification and immunity genes are up-regulated in the hsp-6(mg585) mutant but are not up-regulated in the hsp-6(mg585); nhr-45(mg641) mutant (Fig. 2D), suggesting that these genes are regulated by NHR-45. In order to test whether ARIP-4 participates in the regulation of these genes, we tested two detoxification genes cyp-14A4 and ugt-8 and two immunity genes clec-144 and cnc-9. Their expression levels were examined by quantitative reverse transcription PCR (RT-qPCR). All four genes were up-regulated 10-30 fold in the hsp-6(mg585) mitochondrial mutant, and the induction was decreased more than 70% in either the nhr- 45(mg641) or the arip-4(mg697) mutant (Fig. 2E). Thus, ARIP-4 is a transcriptional coactivator for NHR-45 to promote the activation of detoxification and immune responses, and these proteins move from the nuclear periphery to intranuclear in a coordinated manner upon mitochondrial dysfunction.
Even though mitochondrial stress-induced detoxification and immune responses required UPR mt signaling, loss of nhr-45 had no effect on mitochondrial homeostasis, for example, activation of hsp-6 gene expression (23). We tested the mRNA level of hsp-6 by RT-qPCR, and the up-regulated expression of hsp-6 in hsp-6(mg585) mutant was not affected by either nhr-45(mg641) or arip-4(mg697) (Fig. 2E), which indicates that ARIP-4, like its binding partner NHR-45, does not act in the hsp-6 pathway to promote mitochondrial homeostasis.
As a DNA helicase, we expected that the ATPase activity of ARIP-4 would be essential for its function in regulating transcription. The ATPase activity of ARIP4 was disrupted with a lysine-toalanine mutation at the amino acid 310 (K310A), the ATP-binding site (33). The ATP-binding region of C. elegans ARIP-4 is conserved, and the corresponding lysine in ARIP-4 and the K296A substitution was engineered (Fig. 2F). The function of wild-type ARIP-4 or ARIP-4(K296A) was assayed by the induction of cyp-14A4p::gfp. Wild-type ARIP-4 was able to rescue the arip-4(mg697) mutant and could mediate the induction of cyp-14A4p::gfp caused by hsp-6(mg585); to our surprise, the ARIP-4(K296A) mutant was also able to rescue the arip-4(mg697) mutant and allow induction of cyp-14A4p::gfp (Fig. 2 G and H). Thus, intestinal expression of arip-4 is able to rescue the failure to activate cyp-14A4p::gfp in the hsp-6(mg585); arip-4(mg697) double mutant, suggesting that ARIP-4 regulation of cyp-14A4 is autonomous to the intestine. But, surprisingly, ARIP-4 regulated detoxification and immune responses are independent of its DNA helicase activity.

ARIP-4 Mediates Response to Bacterial Toxins that Attack the
Mitochondrion. The C. elegans NHR-45/MDT-15 pathway mediates detoxification of mitochondrial toxins, for example, the Streptomyces mitotoxin antimycin or the pathogenic P. aeruginosa (23). As the transcriptional coactivator of NHR-45, we tested whether ARIP-4 also acts in this drug and pathogen resistance pathway. The toxicity of antimycin to wild-type animals was established by treating L4 larvae with varying doses of antimycin. Wild-type animals were able to survive as high as 6.25 μg/ml antimycin without significant mortality, but larger doses of 12.5 or 25 μg/ml inhibited growth and survival (Fig. 4A). When L4 larvae of wild-type or arip-4(mg697) animals were treated with 6.25 μg/ml antimycin, the sensitivity of the arip-4(mg697) mutant was dramatically increased relative to wild type (Fig. 4B), indicating the critical role of ARIP-4 in the mitochondrial toxin detoxification and response pathway.
P. aeruginosa produces chemical toxins that disrupt the mitochondrial membrane potential and virulence factors that inhibit translation (27,28). The pathogenic response to P. aeruginosa was examined by growing C. elegans with P. aeruginosa as the sole bacterial food. Compared with the wild-type animals, the arip-4(mg697) mutants were more susceptible to P. aeruginosa (Fig.  4C), suggesting that like its binding partner NHR-45, ARIP-4 acts in the pathway for resistance to P. aeruginosa mitochondrial toxins. While intestine-specific expression of ARIP-4 is sufficient to allow the induction of cyp-14A4 responses to mitochondrial dysfunction, we have not tested whether intestine-specific ARIP-4 expression (Fig. 2 G and H) can rescue the mitochondrial toxin or bacterial pathogen sensitivity of the ARIP-4 mutant (Fig. 4).

Loss of arip-4 Extends C. elegans Healthy Life Span.
Although the detoxification and immune responses defend the mitochondrion from external insults, such as toxins or pathogens, if the mitochondrial dysfunction is caused by a mutation in a nuclearencoded or mitochondrially encoded mitochondrial gene, such responses are futile and might actually compromise health and shorten life span (23). Indeed, loss of nhr-45 improves health status in aged animals and increases life span of the hsp-6(mg585) mitochondrial mutant (23). Therefore, we tested whether decoupling mitochondrial response pathways with an arip-4 mutation might actually increase healthy life span.
The health status of each C. elegans genotype was assessed by the relative speed of movement of young (day 1 adults) or aged (day 10 adults) animals. Wild-type animals move at approximately 200 μm/s on day 1, but by day 10, they move 3× more slowly at about 60 μm/s. The movement speed of hsp-6(mg585) mitochondrial mutant animals was 59 μm/s at day 1, 30% that of wild type, as would be expected for a mutation that affects mitochondrial production of ATP. At day 10, hsp-6(mg585) mitochondrial mutant animals continued to show about 1/3 the motility of wild type with about 20 μm/s (Fig. 5A). Compared with wild type, arip-4(mg697) did not affect locomotion at day 1 but moved at double the speed (128 μm/s) at day 10 ( Fig. 5A). An arip-4 null mutation attenuated the locomotion defects caused by hsp-6(mg585) with speed of movement increased 49% on day 1 and 150% on day 10 (Fig. 5A).
We suggest that the dramatic increase in health span of arip-4; hsp-6 mitochondrial mutants is caused by the decoupling of the impotent detoxification and immune responses to mitochondrial dysfunction in the arip-4(mg697) mutant to render the animals healthier. For example, cytochrome p450 proteins are heme proteins that require iron, often in limiting supply. The nearly 100× induction of cyp-14A4 caused by mitochondrial dysfunction may usurp iron from mitochondrial biogenesis, for example. Similarly, secreted antibacterial proteins pass through the endoplasmic reticulum where multiple cysteines are oxidized to disulfides, an energetically expensive modification. Animals bearing mitochondrial mutations that do not mount impotent but stressful detoxification and immune responses because of the second arip-4 mutation paradoxically fare better. Consistent with this, moderate mitochondrial disruption caused by hypomorphic mutations of essential mitochondrial genes or gene inactivations of nonessential mitochondrial genes extends life span (4,42). For example, the median survival of hsp-6(mg585) (21 d) was substantially increased compared with that of wild type (17 d) (Fig. 5B). While the arip-4(mg697) single mutant, with a median survival of 18 d, marginally increased the life span compared with wild type, the hsp-6(mg585); arip-4(mg697) double mutant lived much longer (24 d) than hsp-6(mg585) (Fig. 5B), showing that the futile arip-4-mediated detoxification response to hsp-6 mitochondrial dysfunction actually shortens life span.
It was notable that the mortality of arip-4(mg697) was extremely low before day 17 (Fig. 5B), which was consistent with the improved locomotion at day 10 (Fig. 5A). The combined analyses of locomotion and life span indicate that loss of arip-4, similar to its binding partner nhr-45, extends healthy life span in wild type and in the hsp-6(mg585) mutant background, emphasizing the deleterious effects of impotent pathogen defense responses triggered by mutations that cannot be detoxified.

Discussion
Disturbance of mitochondrial function by toxins, pathogens, or mutations triggers drug detoxification and immune responses through MDT-15 and NHR-45 mediated transcriptional regulation (23). Our genetic screen for mutants that fail to induce detoxification genes in the hsp-6(mg585) mitochondrial mutant, which identified the nhr-45 mutant, also revealed the arip-4(mg691), a nonsense mutation in arip-4 ( Fig. 1 A and B). C. elegans arip-4 encodes a conserved Snf2-like DNA-dependent ATPase that is orthologous to human ARIP4 (androgen receptor-interacting protein), which also interacts with a NHR, the androgen receptor. We showed that tagged ARIP-4 and NHR-45 proteins colocalize in the nucleus to coregulate cyp-14A4 as well as other genes involved in drug detoxification and antibacterial immunity and that ARIP-4 protein physically interacts with the NHR-45 protein in a similar manner that its human ortholog ARIP4 binds to the NHRs, such as the androgen receptor (35). However, ARIP-4 does not regulate other pathways in mitochondrial homeostasis, such as the gene expression of the mitochondrial chaperone hsp-6, which is consistent with our previous analysis of NHR -45 (23). This response allows C. elegans to detoxify a mitochondrial toxin antimycin and pathogenic P. aeruginosa; loss of this detoxification response in the mdt-15, nhr-45, or arip-4 mutants causes sensitivity to antimycin and P. aeruginosa. However, this detoxification response becomes deleterious if the mitochondrial defect is a mutation that cannot be neutralized by cytochrome p450 induction or other immune responses.
NHR-45 is one of the explosion of novel NHRs in the C. elegans clade with 284 NHR genes compared with humans with 48 NHR genes (43). NHR genes generally show strong conservation in their Zn-finger DNA-binding domains and less conservation in their ligand-binding domains. A comparison of NHR-45 between divergent parasitic nematodes and more closely Caenorhabditae shows that the NHR-45 ligand-binding domain region (aa 300-500) is also conserved, suggesting a conserved small-molecule ligand. An interesting possibility is that the ligand-binding domain of NHR-45 detects a small molecule such as a particular lipid unique to the mitochondrion that is released by the distressed mitochondria to signal mitochondrial dysfunction. In favor of the model that the NHR-45 ligand-binding domain has an actual ligand, NHR-45 ligand-binding domain detects strong homology with the ligand-binding domains of NHR-86, NHR-102, NHR-142, NHR-178, and NHR-213. These NHR proteins form a clade of related NHR genes that may be regulated by related ligands. A mutation in NHR-86 disrupts the induction of the irg-4,5, 6 pathogen response genes by 2-N-(3-chloro-4-methylphenyl)quinazoline-2,4-diamine, which may be a ligand of NHR-86 (44,45). Because such mitochondrial lipid signature molecules may also be synthesized by some species of bacteria, which are ancestrally related to the eukaryotic mitochondrion, NHR-45 may respond to bacterial pathogens as well. Even though NHR-45 has no ortholog in humans, two identified NHR-45 coactivators, ARIP-4/ARIP4 and MDT-15/MED15, are conserved to humans and interact with NHR proteins (37).  and vha-6::arip-4(K296A)::mscarlet, the plasmid was injected into unc-119(ed3) following the miniMOS protocol (38). For CRISPR of arip-4(mg697), we chose dpy-10(cn64) as the co-CRISPR marker (46) and pJW1285 (Addgene) to express both guide RNA (gRNA) and Cas9 enzyme (47).
Immunoprecipitation and Western Blot. Around 20,000 worms of each genotype were synchronized by bleach preparation to L1 larvae, grown to the L4 stage, collected, and frozen by liquid nitrogen. Worm lysates were generated by the TissueLyser with steel beads (Qiagen, 69989) and resuspended in 500 μl lysis buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 1% Triton X-100, and 1X protease inhibitor cocktail). The lysate was centrifuged at 1,000 g for 10 min at 4°C to remove the pellet debris. And the supernatant was divided into two parts: 30 μl to mix with the NuPAGE™ LDS sample buffer (Thermo Fisher, NP0007) as the input and 450 μl to mix with 20 μl GFP-nAb magnetic agarose (Allele Biotechnology, ABP-NAB-GFPX025), which was pretreated twice by the washing buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.5 mM EDTA). The mixture was rotated for 2 h at 4°C to allow the binding. The beads were washed three times with the washing buffer and resuspended in 50 μl 1X NuPAGE™ LDS sample buffer. The input and the beads were heated at 70°C for 10 min. Samples were loaded onto the NuPAGE™ 4 to 12% Bis-Tris protein gels (Thermo Fisher, NP0323BOX) and run with the NuPAGE™ MES SDS running buffer (Thermo Fisher, NP0002). After semidry transfer, the PVDF membrane (Millipore, IPVH00010) was blocked with 5% nonfat milk and probed with anti-GFP (Fisher Scientific, NC9777966) or anti-FLAG (Sigma, F1804) primary antibody and goat anti-mouse IgG HRP secondary antibody (Thermo Fisher, 31430). The membrane was developed with the SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo Fisher, 34096) and visualized by the Amersham Hyperfilm (GE Healthcare, 28906845).
Antimycin Treatment. For each experiment, 30 L4 larvae were treated with different concentrations of antimycin A (Sigma, A8674), and each experiment was repeated three times. To determine the sensitivity of wild-type animals, a twofold serial dilution was performed to antimycin starting from 25 μg/ml. For comparing the sensitivity of wild-type and arip-4(mg697) animals, 6.25 μg/ml antimycin was used. Survival was examined on a daily basis, and the survival curve was generated by GraphPad Prism.
P. aeruginosa Pathogenic Assay. P. aeruginosa PA14 was grown at 37°C overnight, seeded on the NGM plates, and incubated at 37°C for another night. One hundred L4 larvae were picked onto P. aeruginosa lawn and grown at 25°C. Survival was examined on a daily basis, and the survival curve was generated by GraphPad Prism.
Examination of Locomotion. Fifty L4 larvae were picked and grown at 20°C for 10 d. On day 1 and day 10, worms were picked onto bacteria-free NGM plates and photographed directly by the Zeiss AX10 Zoom.V16 microscope. The movement of worms was recorded by continuing picturing every 0.5 s for 30 s in total. The speed of movement was analyzed and calculated by the AxioVision (Zeiss).
Life Span Analysis. Animals were synchronized by egg laying and grown until the L4 stage as day 0. Adults were separated from their progenies by manually transferring to new plates. Survival was examined on a daily basis, and the survival curve was generated by GraphPad Prism.
Data, Materials, and Software Availability. All study data are included in the article.